PROJECT SUMMARY The molecular mechanisms that underpin the development of insulin resistance remain poorly defined, yet a better understanding of this process is critical, given the fact that obesity, metabolic disease and diabetes have reached epidemic status in the US. We discovered that alternative splicing of the insulin receptor, in worms, mice and humans, generates truncated isoforms that are able to attenuate insulin signaling, by inhibiting signaling through the full length receptor. Determination of the physiological significance of these new isoforms is critical in establishing their role in the pathophysiology of insulin resistance. However, such studies in mammals are expensive, time consuming and risky. In contrast, the nematode C. elegans provides an economical system in which to rapidly determine their physiological relevance. To this end, we have started to characterize the truncated isoform of the insulin receptor in the nematode C. elegans, termed DAF-2B, that is directly analogous to mammalian truncated receptors, termed IR-C and IR-D. Preliminary analysis indicates that daf-2b expression varies across tissues and through development. Importantly, we find that genetic deletion of the truncated daf-2b isoform confers insulin sensitivity, while over- expression of a daf-2b cDNA in worms produces phenotypes consistent with attenuated insulin signaling. We are now focused on determining the mechanism(s) by which daf-2b mediates its effects on insulin sensitivity, using a combination of novel reporters that inform on daf-2b dynamics, as well as tissue-specific genetic manipulation of daf-2b. Using a novel reporter strain that permits visualization of differential splicing between the full length and truncated daf-2 isoforms in vivo, we identified specific splicing factors that alter the expression of daf-2b. Exploration of the mechanism by which splicing factor activity regulates the expression of daf-2b will identify novel points of regulation and intervention in this pathway. These discoveries in C. elegans are now guiding studies of the mammalian truncated IRs, including characterization the mode of action of IR-C and IR-D, as well as examining the regulatory factors that lead to expression of these truncated receptors in mammals. Finally, a comprehensive survey of the expression of murine IR-C and IR-D in vivo, relative to full length insulin receptors, will establish their physiological and pathophysiological regulation. We hypothesize that aberrant or mis-regulated expression of truncated IR-C and IR-D isoforms in mammals could be causally involved in the pathogenesis of insulin resistance, diabetes and other forms of metabolic disease. In this respect, the identification of genetic targets and regulatory mechanisms that influence expression of truncated insulin receptor isoforms will expedite the discovery of therapeutic strategies that target this novel insulin regulatory mechanism.